In this section we discuss the conventional Pap test, recent improvements of Pap test, and our preliminary studies that led to discovery and development of the invention.
The Pap test and New Medic al Devices (UDA Approved and Approval Pending) for Improvement of the Pap test.
Pap Test
Pap test is the most used and probably the most successful cancer prevention measure currently available (7). It is recommended for prophylaxis of healthy (asymptomatic) women (8).
Before systematic administration of Pap test, cervical cancer was among the leading causes of death in women with malignant disease. Pap test screening of healthy or oligosymptomatic women resulted in sharp reduction of cervical cancer incidence and mortality rates. Reported are reductions of 80% (Iceland), 70% (U.S.), 50% (Finland) and 34% (Sweden). Recent WHO reports cite that about 4 of every 5 cases of cervical cancer occurs in those countries without screening programs. (11).
TABLE 3 ______________________________________ CLASSIFICATION SYSTEMS FOR PAPANICOLAOU SMEARS Numer- Dysplasia Bethesda CAP ical (cytological) CIN System Score ______________________________________ 1 Benign Benign Normal &lt;100 2 Benign with Benign Normal-Benign- &gt;100 + inflammation with Infection-Reactive inflam- inflam- ASCUS/AGCUS matory mation cells 3 Mild dysplasia CIN I Low grade SIL* &gt;150 3 Moderate dysplasia CIN II ASCUS/AGCUS TBD** High grade SIL TBD 3 Severe dysplasia CIN III TBD 4 Carcinoma in situ TBD 5 Invasive cancer Invasive Invasive cancer &gt;350 cancer ______________________________________ *SIL = squamous intraepithelial lesion. **TBD = to be determined. CAP Score = cervical acid phosphatase activity (our preliminary unpublished results). ASCUS = atypical squamous cells of undetermined significance; AGCUS = atypical glandular cells of undetermined significance.
These positive trends encouraged some of leading workers in the field of cancer prevention to believe that cervical cancer is a curable disease, and that we already have tools for its eradication in the beginning of the 21.sup.st Century. The major obstacle for reaching this ultimate goal of every disease prevention, is the high rate of false negative readings of the Pap test, during the first (primary) screening.
In 1996, an NIH Consensus Conference on Cervical Cancer revealed that 20% of women with a single negative Pap test, developed cervical cancer in the next five years (7). The Conference recommended to increase frequency of screening (annually), to improve sampling (specimen acquisition) techniques, and to improve staining and interpretation (The Bethesda System Classification). These recommendations were widely accepted.(a).
Indeed, many of the current screening protocols call for every women to repeat the Pap test at least once every year for the next five years. It is estimated, if healthy women comply with this schedule, they would have very low probability (0.1%) to develop cervical cancer within this period. However, the vast majority of women do not comply. Another reason for false negatives, is inherent to the Pap test itself Sampling error (omission to bring to the microscopic slides the abnormal cells otherwise present in vaginal fluids or cervical mucosa), and technical error (omission to detect abnormal cells present on smears) are under thorough investigation, and much effort has been given to improve both techniques.(7)
Recent Improvements of the Pap Test
The NIH Consensus Conference's call for Pap test improvement was followed by major development in medical devices industry. Many new patents and instruments have been developed to improve the accuracy of the conventional Pap test. The new technologies are roughly classified into three categories:
FDA approved medical devices related to improvement of the Pap test.
Recently, the FDA has approved three technologies, all users of automatic assistance, that claim reduction of false negative readings as their advantage toward the conventional Pap test.
1) ThinPrep Pap Test.RTM. (Cytyc, Inc.)
This is a liquid sampling technique. This method is intended to replace the Pap test (only in the Sample preparation phase). After the approval, it has been accepted by the HealthCare Corp. In this technology, a physician collects cervical sample in the usual manner, but rather than smearing a small portion of the cervical cells into a slide, a collection device is rinsed in a vial of preservative solution, capturing virtually all of the cell sample. ThinPrep is instrument that disperses and filters the specimen to reduce blood, mucus and inflammation layer of cells to a microscopic slide. ThinPrep 2000 Processor is offered for diagnosis of cervical cancer, lung, bladder, gastrointestinal cancer, and for fine needle biopsy of thyroid and breast cancer.(15)
Disadvantage of this method is that the final sample does not represent the original sample obtained from a patient. Many "inflammatory" and other cells, removed by this cleaning technology could contribute for diagnosis of the body condition. Many small cancer cells may also be lost during the procedure. Implementation needs additional training of Pap test providers, and cytotechnologists (probably cytopathologists, too), and requires buying new equipment, the ThinPrep 2000 Processor. In borderline cases, other techniques are needed to improve sensitivity (i.e., Digene's Hybrid Capture HPV test). The use of this technology adds about $30.00 to the cost of the conventional Pap test.
2) PapNet.RTM. (Neuromedical Systems Inc., NY) (www.nsix.com).
The technology is approved by the FDA for quality control (QC) of the conventional Pap test. All "negative" slides are re-screened by an automated microscope with digital video output. Each cell and cell cluster is analyzed using neural network processing (NNP). NNP is a form of artificial intelligence that has the capacity to "learn," "recognize" and "generalize." Instrument selects 128 cells/clusters for senior cytotechnologist to review "atypical" images. PapNet is particularly good for detection of small atypical cells of high grade lesions that mimic inflammatory cells (16). The cost is $30.00 over the conventional Pap test cost.
Disadvantage of this system is a need for human (high qualified cytologist) interaction after the instrument selected 128 "characteristic" fields. A problem arises during an assessment procedure. This instrument takes images at a single focus and magnification. This is an oversimplification of a microscope as an input device. When a human operator investigates cervical smears using microscopy, he/she always has option to increase magnification and change focus in order to clarify what he/she is seeing and to add more certainty into classification of findings. This is a 3D observation providing more information than any of 2D images or prints that are available to a cytologist interacting with images of microscopic fields already selected by a computer. Computer created images do not have the advantage of human selection.
3) AutoPap.RTM. (NeoPath, Inc., WA)(www.neopath.com)
AutoPap was approved by the FDA for quality control screening of "negative" Pap smears. Recently, this system was also approved for the primary screening (new arrivals) of Pap smears.
According to the sponsor, the NeoPath, Inc., the AutoPap Primary Screening System is an automated cervical cytology screening device intended for use in initial screening of Papanicolaou (Pap) smear slides. The device is to be used only on conventionally prepared Pap smear slides and is intended to detect slides with evidence of squamous carcinoma and adenocarcinoma and their usual precursor conditions; it is not intended to be used on slides designated by the laboratory as "high risk." Intended users are trained cytology laboratory personnel operating under the direct supervision of a qualified cytology supervisor or laboratory manager/director. The cost is $20.00 above the cost of the conventional Pap test. (11,11a).
Disadvantage of this method is that it still needs a qualified cytologist to review "negative" slides, and limitation of disease conditions.
4) Accustain.RTM. Papanicolaou Staining System (Sigma Chem. Co., St. Louis, Mo.). Procedure HT40 (1994) for research purpose only. (52)
The original Pap test has never been patented. Stains used in the Papanicolaou staining cannot be patented as stains. Instructions for Pap smear staining are vague and permit individual laboratories to adjust dye concentrations and staining time according to requirement of local cytopathologists. Staining characteristics differ from lab to lab and external quality control is difficult to obtain. Accustain is a kit. It was introduced with intention to standardize the Pap staining. We are following the same idea--our kit will be standardized and will provide more staining uniformity than it is currently available. This achievement alone could contribute for reduction of false negative readings.
FDA approval pending medical devices Several systems are still in development.
1) LSC.RTM. (Laser Scanning Cytometer, CompuCyte, Corp. MA).
This instrument combines light microscopy, scanning stage, image analysis and multiple staining procedures. This combination was made possible by a strong control of the scanning stage and location of cells. However, staining procedures are subsequent each to other is time consuming, and many artifacts could occur (17).
Human interaction is necessary. (www.compucyte.com).
2) AcCell.TM.Cytopathology System (AccuMed International, Inc., Chicago, Ill.).
This is a computerized microscope workstation. TracCell.TM. is a workstation software that integrates specimen automated stage. The system is intended to determine coordinates of areas of "no interest" and to give cytologist only areas "of interest" for observation. Indeed, this approach increases the possibility for misreading of smears (cytologist sees only fraction of the smear).(www.accumed.com)(18)
3) AutoCytePrep and AutoCyteScreen (AutoCyte, Inc. NC).
This one of liquid collection oriented methods, which is advertized as a non-gyn test. Both have improved cell collection and slide/smear preparation yielding to multiple staining (in succession) and more advanced image analysis. An interesting instrument, but needs human interaction for screening.(www.autocyte.com)(19)
4) Hybrid Capture.RTM.HPV Test (Digene Corp., MD).
The only approved system for detection of HPV in cervical smears. Reeds women's DNA to identify HPV (infection that may contribute to cervical cancer). It is always perform ed together with the Pap test for morphological determination.(www.digene.com). Can use ThinPrep solution for specimen collection.(20)
5) DiaDexus Joint Venture.
SmithKline Beacham, PLC, and Incyte Pharmaceutical Inc., have recently (December 1998) announced a joint venture "DiaDexus" to study a new test for improvement of the conventional Pap test. Their test uses "antibodies to home in on any abnormal cells in a cervical smear", which could enable them to serve as markers for "replicating cells that are potentially cancerous." Naturally, clinical trials will give the answer. However, it is known that not only cancerous cells replicate, but many other cells, including normal cervical epithelial cells do so. More information is necessary before conclusion on this "new test."(21)
For more information on diagnostics devices available for cervical cancer screening a reader should refer to the recent review articles (22).
Regulatory Issues
All devices described above qualify for category III medical devices, meaning they need to be at least equivalent (safety and efficacy) to an approved device (method) already on the market. However, the "golden standard" to measure safety and efficacy of Pap test related devices is the conventional Pap test itself. And this test has never been standardized or approved as a single in vitro diagnostic device.
A common practice is to use a meta-analysis of data from several cytopathology laboratories using the unstandardized, conventional Pap test, and to create a historic group to be a comparator for the new device. Such a historic group is a very weak comparator for statistical analyses of equivalence. On the other side, all devices described above, have been designed rather as adjunct than a substitute for the conventional Pap test. Therefore, it is very unlikely, they could be able to sustain a scrutiny of statistical analyses in clinical trials (unless with a very large sample size).
We have designed clinical trials to show superiority of CAP-PAP Test versus Pap test to detect cervical dysplasia (and/or cancer). Two of them are competing for SBIR Grants:
Our expectation is that our product, a well standardized and controlled kit, will replace the conventional Pap test.
Summary on Pap test
Clinical trials testing superiority of new technologies vs the conventional Pap test, either have not been completed, or have not demonstrated significant (or trend) superiority. However, they all have shown an increased cost. Recently (April 1999) Medicare reimbursed $7.15 for the Pap test. A proposed legislation tries to limit the lowest reimbursement rate above $14.60 (25). Higher cost per individual test is an inherent problem of all currently available technologies cited above. If implemented, they could significantly (from 10 to 40 dollars a test) increase the cost of primary screening for cervical cancer. With about 80 millions tests per year, it is an extremely expensive task to go. It is not likely the Government (Medicare) or other health insurance companies will proceed using these services. It is more likely, they will continue to search for another, less expensive methods for improvement of th e Pap test,
In conclusion, the quest for a reliable, rapid and inexpensive technology that could help eradicate cervical cancer in the next Century, has not been completed yet. We
Cervical Acid Phosphatase--Overview of the Prior Art
Introduction.
Acid phosphatases are ubiquitary enzymes. They are found in plants and animals. In humans they have been intensely investigated in prostate, liver, kidney and connective tissue, particularly blood cells.(26-29). As enzymes, they all release phosphate from organophosphate. With regard to substrate preference, they have shown species and tissue specificity.
Demonstration of acid phosphatase in tissues and cells is based on enzyme catalysis of organophosphate substrate, caption of phosphate by a metallic ion (i.e. lead), or an organic radical (aromatic ring) by a diazonium salt, formation of a product which is insoluble at acid pH range (pH&lt;5.0), and precipitation of a colorful, granular deposit at sites of enzyme activity (FRP). FRP is available for microscopic examination. The amount is measurable and it is proportional to acid phosphatase activity. (28,30).
Many human cell types and tissues contain acid phosphatase. In humans, acid phosphatase is confined inside lysosome.
Acid phosphatase is abundant in metabolically active cells in inflammation and malignancy.
Medical literature contains many data related to acid phosphatase activity in different human cells and tissues (31-35). Acquired knowledge is helpful as guidance for understanding of the mechanisms involved in the cervical acid phosphatase testing. The alteration of synthesis, processing and trafficking of lysosomal enzymes in malignancy has been demonstrated (36-38). A consistent increase of lysosomal enzymes (i.e., prostatic acid phosphatase) has been found in tumor cells in comparison with their normal counterparts (29,39,40). This property contributes to "aggressiveness" of malignant cells. In blood cells, an increase of acid phosphatase activity was found in connection with infection and inflammation (i.e., PMN, monocytes). Further reference on this matter can be found in review articles (22).
There are many studies in forensic medicine that are all related to demonstration of semen acid phosphatase in vaginal fluid as evidence for sexual intercourse. Information of acid phosphatase activity in cervical epithelial cells is scarcely.
Cervical Acid Phosphatase--Clinical Experience
In healthy women, normal looking cervical epithelial cells contain alkaline phosphatase. Acid phosphatase has been described rarely if it has been described at all. However, acid phosphatase in vaginal fluid/smears has been suited intensely in forensic medicine as indicator of rape (enzyme from semen). (41).
Medical literature contains only few articles related to cervical acid phosphatase. In 1960, Gross and Kinzie found the gradient of acid phosphatase activity in malignant epithelium to be similar to the normal cervical epithelium; however poorly differentiated, malignant cells had a higher degree of activity (42). They used a Gomori's method for visualization of cervical acid phosphatase. In 1961 Berger showed semiquantitative difference between acid phosphatase activity in basal and malignant cells. Mature cervical epithelial cells did not present that type of activity (43).
In 1974, Malvi et al., described acid phosphatase in carcinoma of the cervix uteri (44). Using a staining technique according to Gomori, they found increased enzyme activity in malignant cells as opposing to "normal" activity in basal cells.
In 1978, Panazzolo et al. (46) using the same Gomori's method described acid phosphatase-positive granulations in epithelial cells originating from in the vaginal secretion of 44 patients suffering from cervical (17 cases) and uterine cancer (27 cases). They found a highly increased acid phosphatase activity in preinvasive forms, and less activity in invasive cancers. Discussing data they said:
It was only discussion (speculation) based on no data. We were not able to find any follow-up to this study.
Our studies confirmed these old data: cervical acid phosphatase can be found only in abnormal cervical epithelial cells.
Preliminary study: Development of the CAP-PAP T est.
In 1986-88, we were investigating a number of human tissue specimens versus many cytochemical techniques to select candidates for quantitative image analysis. In one of series, cervical smears were exposed to cytochemical techniques for demonstration of lysosomal enzymes. Surprisingly, acid phosphatase activity was found in atypical squamous epithelial cells while normal-looking cells did not present this type of activity.(47). We used an azo dye diazonium salt technique. Reviewing literature we found only few articles. The authors, using Gomori's method have come to similar findings. It was obvious that we were the first to demonstrate acid phosphatase activity inside abnormal cervical cells using a diazonium dye staining technique. (1,2,48).
Pursuing this finding, we initiated and designed two studies. Because of various reasons, none of them was completed or published. For the purpose of this patent application we describe here some of crucial findings from those studies.
1987/88 Study: Method for Visualization of Cervical Acid Phosphatase
In 1987/88 we used a double-slide, single-staining technique. It was a small study. Fifty randomly selected women with some kind of pelvic disorders agreed to allow doctors to use their Pap specimen and to smear the spare material onto an additional slide. Two unfixed smears were prepared in doctor's office and sent to laboratory. Within few hours, one of them was stained by Papanicolaou technique (control). The other was stained for acid phosphatase (test). We used a Sigma procedure for staining leukocyte acid phosphatase Cat. No. 387-A and 386-A)
Both smears were independently, but open-label, reviewed by a cytopathologist, a cytochemist and a hematologist. We compared both smears using morphologic criteria for detection of cervical intraepithelial neoplasms (C,N classification). Our conclusions are summarized on the following table.
TABLE XX ______________________________________ Cervical Acid Phosphatase, CIN Classification of Cervical Smears, and Cervical Epithelial Cell Type (Study: CAP-HUP-1986) Dominant cell type Cell type CAP CIN &gt;85% of cells distribution Cell characteristics Result ______________________________________ 0 LCC L Large epithelial cell, 0, + abundant clear cytoplasm, small nucleus, condensed chromatin 1 MCC L &gt; M &gt; S Moderate size epithelial +, ++ 2 M &gt; S &gt; L cell, abundant cytoplasm, larger nucleus, nucleolus absent, chromatin pattern dense 3 SCC S &gt; M &gt; L Small epithelial cell, ++, +++ C/N = 1.5 and above, large nucleus, nucleoli present ______________________________________ CC = cervical cells; L = large, M = intermediate; S = small
1996-97 Study: Conceiving the Idea
In 1996 the NIH Consensus Statement on Cervical Cancer revealed that 20% of women with a single negative single Pap test developed cervical cancer during the following five years. Among other recommendations, the Consensus Conference emphasized needs for technical improvement of the Pap test technology. (7).
We thought we have a new marker which seemed specific for abnormal cervical epithelial cells. Would it be the answer to the problem? To repeat our old findings we designed a small study. Fifteen women, referring to a doctor's office for pelvic exam or regular Pap smear, consented that spare parts of their Pap specimen be used for research purpose. Spare smears were exposed to the CAP procedure (double-slide, single-staining). The results confirmed our observations from 1988: acid phosphatase activity was inversely proportional to maturity of epithelial cells, and abnormal-looking cells possessed most active acid phosphatase.
A double-staining, single-slide method: The CAP-PAP Test.
At this time we have realized that the double-slide, single-staining procedure is not for a routine field application. Therefore we have concentrated our attention to develop a procedure that will provide the benefit of an acid phosphatase marker on the same slide where cells are stained by Papanicolaou staining. The idea was to add a new marker of abnormality to the classical morphologic criteria for cervical cancer screening. To achieve this goal we used HeLa cell line as a breadboard for our experiments. This a human cervical carcinoma derived cell line. All cells contain acid phosphatase activity. We exposed smears of these cells to different cytochemical and Papanicolaou staining conditions. Finally we achieved conditions that could provide simultaneous staining of the marker and cell morphology. The double-staining, single-slide procedure was born. In a follow-up, we applied the new procedure to spare cervical smears. The result confirmed our expectations: acid phosphatase could be stained inside abnormal cervical cells, and counterstained by a modified Papanicolaou (for visualization of classical cytologic criteria). Normal cervical cells did not stain for acid phosphatase. (50, 51). This procedure was named the Cervical Acid Phosphatase-Papanicolaou (CAP-PAP) Test.
The discovery of a new method was followed by an Invention Disclosure (USPTO #426850 of Oct. 24, 1997)(1), and the Provisional Patent Application (USPTO #60/09744 of Aug. 14, 1998)(2).
In 1998, we applied for SBIR grants. The application was under the Fast Track Initiative and included two clinical trials (BSC-98-02 phase I/I, and BSC-98-03, phase II)(23,24). Due to administrative deficiencies (lack of Business Plan) this application was transferred to a Phase 1 SBIR grant procedure and did not receive funds in this round (Dec. 15, 1998 deadline). We intent to correct deficiencies and to resubmit the application in the first round of 1999.